JP2017000086A - Novel peptidase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel peptidase suitable for use in the field of food production or the like.SOLUTION: The peptide has the following enzymatic chemical properties: (1) action: catalyze the reaction of liberating an amino acid from the N-terminus of a peptide chain; (2) molecular weight: a molecular weight of about 75.3 kDa in SDS-PAGE; (3) substrate specificity: liberate leucine, arginine, methionine, and alanine from the N-terminus of the peptide chain, poor action on glycine and proline at the N-terminus of the peptide chain, and no action on a proline-bound amino acid at the N-terminus; (4) optimum pH: approximately 8; and (5) optimum temperature: approximately 40°C. The peptide having has the following enzymatic chemical properties: (1) action: catalyze the reaction of liberating an amino acid from the C-terminus of a peptide chain; (2) molecular weight: a molecular weight of about 70.5 kDa in SDS-PAGE; (3) substrate specificity: liberate leucine and phenylalanine from the C-terminus of the peptide chain and poor action on alanine, proline, and tyrosine at the C-terminus of the peptide chain; (4) optimum pH: approximately 5; and (5) optimum temperature: approximately 50°C.SELECTED DRAWING: None

Description

  The present invention relates to peptidases. Specifically, it relates to a peptidase derived from Pseudozyma hubeiensis and its use.

  Proteolytic enzymes are roughly classified into endopeptidases (proteinases) and exopeptidases (peptidases) according to their mode of action. Exopeptidases include aminopeptidases that catalyze the release of amino acids from the N-terminus of peptides and carboxypeptidases that catalyze the release of amino acids from the C-terminus. Until now, various peptidases derived from microorganisms are known, and application and application to various uses have been attempted.

  Peptidases have been reported to be isolated from yeast (Non-Patent Documents 1 and 2), filamentous fungi (Non-Patent Documents 3 and 4), bacteria (Non-Patent Documents 5 and 6), and the like. In particular, there are many reports of peptidases derived from Saccharomyces cerevisiae (Non-Patent Documents 7 to 9). Aminopeptidases derived from Saccharomyces cerevisiae include leucylaminopeptidase (EC 3.4.11.1), aminopeptidase Y (EC 3.4.11.15), aminopeptidase I (EC 3.4.11.22), and carboxypeptidase Y (EC). EC 3.4.16.5) is known.

  Peptidases are used in food processing and fermented food production. For example, it is used for enhancing the flavor of seasonings and yogurt, and promoting ripening of cheese.

Moudni, E. B. et al., (1995) J. Med. Microbiol., 43, 282-288. Klionsky, J. D. et al., (L992) J. Cell Biol., 119, 287-299. Nishiwaki, T and Hayashi, K. (2001) Biosci. Biotechnol. Biochem., 65,424-427. Sattar, A. K. M. et al., (1989) Agr. Biol. Chem., 274.241-250. Izawa, N. et al., (1997) J. Agric. Food Chem., 45, 543-545. Story, V. S. et al., (2005) J. Bacteriol. 187, 2077-2083. Matile, P. et al., (1970) Planta, 96, 43-53. Metz, G, and Rohill, K.-H. (1976) Biochem. Biophy. Acta., 429, 933-946. Achstetter, T. et al., (1982) Biophy. Res. Commu., 109,341-347. Nishiwaki, T. et al., (2002) J. Biosci. Bioeng., 93, 60-63.

Aminopeptidases and carboxypeptidases derived from bacteria such as Aspergillus, Rhizopus, or Bacillus are produced on an industrial scale as food additives. However, since proteinases are mixed in these peptidases (compositions), unintended effects are caused by the action of proteinases. For example, the physical properties of food change or a bitter peptide is produced. One of the effects expected of peptidases is the reduction of bitterness due to the degradation of bitter peptides contained in foods and the like (Non-patent Document 10), but the inclusion of proteinases reduces the effects. On the other hand, although peptidase derived from Saccharomyces cerevisiae exerts a bitter taste reducing effect, it is an intracellular enzyme, and therefore, productivity and economy are problems in industrial utilization (eg, food production).
Therefore, an object is to provide a novel peptidase suitable for use in fields such as food production.

The present inventor has repeatedly studied in view of the above problems. As a result, it was clarified that the yeast Pseudozyma hubeiensis 31-B strain isolated from the digestive fluid in the insect trap of Nepenthes alata produced aminopeptidase and carboxypeptidase outside the cells. In addition, these peptidases were successfully purified and their properties were identified. Furthermore, it has been clarified that these peptidases exhibit a degrading activity on bitter peptides and exhibit an excellent bitter taste reducing effect. On the other hand, it was found that Pseudozyma / Fubeyensis 31-B strain hardly produces contaminating proteinase. This feature is a great merit in formulating peptidase.
The following invention is mainly based on the above-mentioned results.
[1] Peptidase having the following enzyme chemical properties:
(1) Action It catalyzes a reaction that liberates the N-terminal amino acid of the peptide chain;
(2) Molecular weight The molecular weight by SDS-PAGE is about 75.3 kDa;
(3) Substrate specificity Releases leucine, arginine, methionine, and alanine at the N-terminus of the peptide chain. The effect on N-terminal glycine and proline of the peptide chain is weak. Does not affect the N-terminal amino acid bound to proline;
(4) optimum pH about 8;
(5) Optimum temperature about 40 ℃.
[2] Peptidase having the following enzymatic chemistry:
(1) Action It catalyzes a reaction that liberates the C-terminal amino acid of the peptide chain;
(2) Molecular weight The molecular weight by SDS-PAGE is about 70.5 kDa;
(3) Substrate specificity It works well on leucine and phenylalanine at the C-terminal of the peptide chain. Weak effect on alanine, proline and tyrosine at the C-terminus of the peptide chain;
(4) optimum pH about 5;
(5) Optimum temperature about 50 ℃.
[3] The peptidase according to [1] or [2], which is derived from Pseudozyma fubayensis.
[4] The peptidase according to [3], wherein Pseudozyma hubeiensis is Pseudozyma hubeiensis 31-B strain.
[5] An enzyme agent comprising the peptidase according to any one of [1] to [4] as an active ingredient.
[6] An enzyme agent comprising the peptidase according to [1] and the peptidase according to [2].
[7] A method for producing a peptidase comprising the following steps (1) and (2):
(1) culturing Pseudozyma huveiensis;
(2) A step of recovering peptidase from the culture solution after culturing.
[8] The production method according to [7], wherein Pseudozyma / Fubeyensis is Pseudozyma / Fubeyensis 31-B strain.
[9] The production method according to [7] or [8], wherein aminopeptidase and carboxypeptidase are recovered as the peptidase in step (2).
[10] The production method according to [7] or [8], wherein aminopeptidase or carboxypeptidase is recovered as the peptidase in step (2).
[11] Pseudozyma hubeiensis 31-B strain specified by the accession number NITE P-02047.
[12] Peptidase according to any one of [1] to [4], or [5] or [6] for a raw material or intermediate processed product containing protein or peptide in the production process of food A method for producing or processing a food, characterized by causing the enzyme agent to act.
[13] The method according to [12], wherein the food is a dairy product.
[14] A food with reduced bitterness due to the action of the peptidase according to any one of [1] to [4] or the enzyme agent according to [5] or [6].

Results of DEAE-Toyopearl 650M chromatography. ◆; protein (280 nm), ■; LAP activity (410 nm), ▲; ACP activity (460 nm). Results of SDS-PAGE (AP31-B). Lane 1 is molecular weight marker, lane 2 is AP31-B. SDS-PAGE result (CP31-B). Lane 1 is CP31-B, Lane 2 is molecular weight marker. Temperature characteristics of carboxypeptidase (CP31-B). Temperature characteristics of aminopeptidase (AP31-B). PH characteristics of carboxypeptidase (CP31-B). ◆; McIlvaine buffer, ■; phosphate buffer, ▲; Tris buffer. PH characteristics of aminopeptidase (AP31-B). ■; McIlvaine buffer, ○; phosphate buffer, ●; Tris buffer, □; Atkins-Pantin buffer. Reduction of bitterness by aminopeptidase (AP31-B). ●: Decomposition rate, ○ bitterness. Reduction of bitterness by carboxypeptidase (CP31-B). ◆: Decomposition rate, ■; Bitterness. Effect of pH on aminopeptidase activity of crude enzyme (PP31). Effect of temperature on aminopeptidase activity of crude enzyme (PP31). Effect of temperature on the carboxypeptidase activity of the crude enzyme (PP31). Bitter taste reduction effect of crude enzyme (PP31) (pH 7.0) Bitterness reduction effect of crude enzyme (PP31) (pH 5.0)

1. Peptidase and its producing bacteria The first aspect of the present invention provides a peptidase and its producing bacteria. As shown in the Examples described later, the present inventors have intensively studied and found that Pseudozyma hubeiensis 31-B strain produces two kinds of characteristic peptidases (aminopeptidase and carboxypeptidase). It was. Moreover, these peptidases were successfully purified and their enzymatic chemistry was determined as shown below. Hereinafter, the enzymatic chemistry of each peptidase will be described.

<Aminopeptidase>
(1) Action This enzyme is an aminopeptidase and catalyzes a reaction that liberates the N-terminal amino acid of the peptide chain.
(2) Molecular weight The molecular weight of this enzyme is about 75.3 kDa (by SDS-PAGE).
(3) Substrate specificity This enzyme releases leucine, arginine, methionine, alanine and the like at the N-terminal of the peptide chain. The effect on N-terminal glycine and proline of the peptide chain is weak. Does not affect the N-terminal amino acid bound to proline.
(4) Optimum pH
The optimum pH of this enzyme is about 8. The enzyme exhibits high activity at a pH of about 6.5 to about 9.5. The LAP method (the temperature during the reaction was 37 ° C.) described later was used for measuring the optimum pH. McIlvaine buffer (pH 4-6), phosphate buffer (pH 6-7), Tris-HCl buffer (pH 7-9), Atkins-Pantin buffer (pH 9-10) were used. .
(5) Optimal temperature The optimal temperature of this enzyme is about 40 ° C. This enzyme exhibits high activity at about 30 ° C to about 55 ° C. The LAP method described later (pH at the time of reaction is 8.0) was used for measuring the optimum temperature.
(6) Other properties The activity is completely inhibited by zinc ions (Zn ²⁺ ), and also by divalent iron ions (Fe ²⁺ ), copper ions (Cu ²⁺ ) and trivalent iron ions (Fe ³⁺ ). Activity is inhibited. The activity is also inhibited by SDS, EDTA and PCMB. The activity is not inhibited by E-64, Pepstatin A and Petabloc. On the other hand, potassium ions (K ⁺ ) promote activation.

<Carboxypeptidase>
(1) Action This enzyme is a carboxypeptidase and catalyzes a reaction that liberates the C-terminal amino acid of the peptide chain.
(2) Molecular weight The molecular weight of this enzyme is about 70.5 kDa (by SDS-PAGE).
(3) Substrate specificity This enzyme works well on C-terminal leucine and phenylalanine of peptide chains. The effect on alanine, proline and tyrosine at the C-terminal of the peptide chain is weak.
(4) Optimum pH
The optimum pH of this enzyme is about 5. The enzyme exhibits high activity at a pH of about 3.0 to about 5.5. The CP method described later (temperature at the time of reaction is 40 ° C.) was used for the measurement of the optimum pH. McIlvaine buffer (pH 3-6), phosphate buffer (pH 6-7), Tris-HCl buffer (pH 7-9) were used.
(5) Optimal temperature The optimal temperature of this enzyme is about 50 ° C. This enzyme exhibits high activity at about 30 ° C to about 55 ° C. The ACP method described later (pH at the time of reaction is 5.0) was used for the measurement of the optimum temperature.
(6) Other characteristics The activity is inhibited by potassium ion (K ⁺ ), manganese ion (Mn ²⁺ ) and divalent iron ion (Fe ²⁺ ). The activity is also inhibited by SDS and PCMB.

  The peptidase of the present invention is preferably an enzyme derived from Pseudozyma huveiensis, more preferably Pseudozyma hubeiensis 31-B. The term “peptidase derived from Pseudozyma / Fubeyensis 31-B” as used herein means peptidase produced by Pseudozyma / Fubeyensis 31-B or Pseudozyma / Fubeyensis 31-B (a wild-type mutant is a mutant strain). It may be a peptidase obtained by genetic engineering techniques using the peptidase gene. Therefore, a recombinant produced by a host microorganism into which a peptidase gene obtained from Pseudozyma / Fubeyensis 31-B (or a gene modified from the gene) is also referred to as “peptidase derived from Pseudozyma / Fubeyensis 31-B”. Applicable.

For convenience of explanation, the Pseudozyma flaviensis 31-B strain from which the peptidase of the present invention is derived is referred to as a peptidase producing bacterium of the present invention. Pseudozyma Hubeiensis 31-B strain is deposited with the prescribed depository as follows and is easily available.
Depositary Institution: National Institute of Technology and Evaluation Biotechnology Center Patent Microorganism Depositary Center (NPMD) (Room 122, 2-5-8 Kazusa Kamashi, Kisarazu, Chiba, 292-0818, Japan)
Deposit date: May 11, 2015 Deposit number: NITE P-02047

2. Enzyme Agent The peptidase of the present invention is provided, for example, in the form of an enzyme agent. In addition to the active ingredient (peptidase), the enzyme agent may contain excipients, buffers, suspension agents, stabilizers, preservatives, preservatives, physiological saline and the like. As the excipient, lactose, sorbitol, D-mannitol, sucrose and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like can be used.

  The enzyme agent of the present invention contains the above aminopeptidase or carboxypeptidase, or both of them as active ingredients. The latter embodiment, that is, an enzyme agent containing both aminopeptidase and carboxypeptidase as active ingredients is characteristic of the present invention. As shown in the Examples described later, as a result of the study by the present inventors, it was revealed that Pseudozyma / Fubeyensis 31-B strain produces the above two enzymes outside the cells. Since it is produced outside the cells, high productivity can be realized. On the other hand, it was also revealed that Pseudozyma / Fubeyensis 31-B strain does not substantially contain proteinase outside the cell. This fact means that it is possible to prepare a peptidase agent containing aminopeptidase and carboxypeptidase as active ingredients substantially free from contaminating proteinases without going through complicated purification steps. That is, formulation is easy. Moreover, even if it refine | purifies, the operation and process for that will become a simple thing.

  An enzyme agent containing both aminopeptidase and carboxypeptidase as active ingredients is obtained by, for example, filtering and centrifuging the culture supernatant of Pseudozyma / Fubeyensis 31-B strain to remove insoluble matters and concentrating it. Can do. Alternatively, the culture supernatant can be purified by one or more purification steps selected from the group consisting of removal of impurities, concentration, dilution, salting out, dialysis, dissolution and drying. Since the culture supernatant of Pseudozyma / Fubeyensis 31-B is substantially free of proteinase, it is substantially free of contaminating proteinase even without a purification step (for example, column chromatography) for removing the proteinase. Thus, a peptidase agent containing aminopeptidase and carboxypeptidase as active ingredients can be prepared.

3. Method for Producing Peptidase A further aspect of the present invention provides a method for producing a peptidase. In the present invention, (1) a step of cultivating Pseudozyma fubeiensis and (2) a step of recovering peptidase from the culture solution after culturing. Isolation and purification may be performed as one aspect of the recovery.

  In step (1), Pseudozyma fubeiensis is cultured. Pseudozyma huveiensis is not particularly limited as long as it has the ability to produce the desired peptidase. For example, the above-mentioned Pseudozyma / Fubeyensis 31-B strain can be used. The culture method and culture conditions are not particularly limited as long as the target enzyme (peptidase) is produced. That is, on the condition that the peptidase of the present invention is produced, a method and culture conditions suitable for culture can be set as appropriate. The culture method may be either liquid culture or solid culture, but preferably liquid culture is used. Taking liquid culture as an example, the culture conditions will be described.

  The medium is not particularly limited as long as it is a medium in which Pseudozyma fubeiensis can grow. For example, glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, carbon sources such as organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein water A medium supplemented with a nitrogen source such as a decomposed product, bran or meat extract, or an inorganic salt such as potassium salt, magnesium salt, sodium salt, phosphate, manganese salt, iron salt or zinc salt can be used. Vitamins, amino acids, and the like may be added to the medium in order to promote the growth of Pseudomasa fubayensis. The pH of the medium is adjusted to, for example, about 4 to 7, preferably about 6 to 7, and the culture temperature is usually about 20 ° C to 35 ° C, preferably about 25 ° C to 30 ° C, preferably 1 day to 5 days. Is cultured under aerobic conditions for about 2 to 3 days. As the culture method, for example, a shaking culture method or an aerobic deep culture method using a jar fermenter can be used.

  After culturing under the above conditions, peptidase is recovered from the culture solution (step (2)). For example, the insoluble matter is removed by filtering, centrifuging, or the like of the culture solution to obtain the target peptidase solution. Further, purification steps such as concentration, dilution, salting out, dialysis, dissolution, and drying may be performed to obtain a peptidase with high purity. According to the method as described above, a composition containing both aminopeptidase and carboxypeptidase can be obtained. On the other hand, by applying various types of chromatography (ion exchange chromatography, adsorption chromatography, hydrophobic chromatography, etc.), solvent fractionation, pH treatment, heat treatment, etc., one of the peptidases is removed and aminopeptidase or carboxypeptidase is removed. May be selectively collected.

  The degree of purification of the recovered peptidase is not particularly limited. For example, the peptidase can be purified to a specific activity of 10 to 500 (U / mg). The final form may be liquid or solid (including powder). The recovered peptidase can be provided by pulverization, for example, by freeze drying, vacuum drying or spray drying. At that time, the purified enzyme may be dissolved in a phosphate buffer, triethanolamine buffer, Tris-HCl buffer or GOOD buffer in advance. Preferably, a phosphate buffer or Tris-HCl buffer can be used. In addition, PIPES, MES, or MOPS is mentioned as a GOOD buffer here.

4). Use of Peptidase A further aspect of the present invention provides a food production / processing method as use of the peptidase. In the method of the present invention, the peptidase of the present invention is allowed to act on the raw material or intermediate processed product during the production or processing of food. Instead of peptidase, an enzyme agent containing it may be used.

  Typically, the peptidase or enzyme agent of the present invention is added to the raw material or intermediate processed product to be treated, and the peptidase is treated. If conditions for the action of the peptidase are formed in the normal production / processing process, it is not necessary to provide a dedicated process for causing the peptidase to act. That is, in one embodiment, the food is manufactured by a normal manufacturing / processing process, except that peptidase is added. Instead of adding the peptidase, the peptidase may be allowed to act by, for example, immersing a raw material of food or an intermediate processed product in a solution containing the peptidase.

  As foods to which the present invention can be applied, dairy products (milk, cheese, yogurt, etc.), meat, processed meat products, meat extracts, fish products, processed fish products, fish extracts, yeast extracts, processed yeast extracts (vegemite, marmite) Etc.), bouillon, consomme, vegetable, processed vegetable product, vegetable extract, fruit, processed fruit product, fruit juice, processed fruit product, cereal, cereal powder, processed cereal product, retort food, cooked food, nutritional supplement (supplement) And fermented foods such as food drinks, pet foods, soy sauce, and miso.

  According to the method of the present invention, a bitter taste peptide is decomposed by the action of peptidase, and a food with reduced bitterness can be obtained. Therefore, the present invention is particularly useful for the production or processing of foods whose bitter taste negatively affects their quality (foods that are preferred to have low bitterness). In addition, it is highly useful in the production or processing of foods and food additives (for example, meat extract, fish extract, yeast extract, etc.) for which bitterness is not preferred due to their use.

  Endopeptidases (proteinases), aminopeptidases, and carboxypeptidases are known as yeast proteolytic enzymes. These proteinases and peptidases are found in the autolysate of yeast cells. Therefore, it is necessary to extract from the microbial cells, and when viewed as an industrial enzyme, the productivity is questionable. To date, there are few reports of peptidases produced outside the cells. Then, the following examination was performed aiming at acquisition of the novel peptidase derived from yeast produced outside a microbial cell.

<Acquisition of new peptidase>
A. Materials and Methods Strain At the Higashiyama Botanical Garden (Nagoya, Japan), it has a high ability to produce exopeptidase without producing proteinase outside the cell from the yeast isolated from the digestive fluid in the insect trapping bag of the nematode Nepenthes alata One strain (31-B strain) was found and used for research.

各種培地組成 (%) 2. Yeast culture
The yeast 31-B strain suspension suspended in 13% glycerin solution and stored at -85 ° C was applied to the slant medium shown in A of Table 1 and cultured at 25 ° C for 2 days. Next, one platinum loop was inoculated from the slant culture into 140 mL of the seed medium placed in a 500 mL shake flask shown in B of Table 1, and cultured with shaking (180 rpm) at 25 ° C. for 24 hours. Subsequently, 140 mL of the seed culture solution was inoculated into a 10 L jar fermenter containing 7 L of the culture solution shown in C of Table 1, and aerated and stirred at 25 ° C (0.5 vvm, 500 rpm, 72 hr) did.

Medium composition (%)

3. Activity measurement method 3-1. Carboxypeptidase activity measurement method 3-1-1. ACP (Acid Carboxypeptidase) Method Using an acid carboxypeptidase measurement kit (Kikkoman Biochemifa Co., Ltd.), the measurement was performed under the reaction conditions of 37 ° C. according to the kit specifications. The activity was displayed according to the kit specifications, and the amount of enzyme that liberates 1 μmol of L-alanine from the substrate Cbz-Tyr-Ala per minute was defined as 1 unit.

3-1-2. CP (carboxypeptidase) method
To 0.9 mL of Z-Phe-Leu (20.6 mg / 0.05M acetate buffer, pH 5.0) solution, 0.1 mL of the enzyme solution was added and reacted at 37 ° C. for 30 minutes. After adding 1 mL of ninhydrin solution, it was left in boiling water for 15 minutes to give color to the released amino acid. After cooling in running water, 5 mL of 50% ethanol solution was added, and after sufficiently stirring, the absorbance at 570 nm was measured. The amount of enzyme that liberates 1 μmol of leucine per minute under the above conditions was defined as 1 unit.

3-2. Aminopeptidase (LAP) activity assay
1 mL of 200 mM Tris-HCl buffer (pH 8.0) was added to 0.8 mL of L-leucyl-p-nitroanilide hydrochloride solution (71.5 mg / 100 mL purified water), and preheated to 37 ° C. for 5 minutes. After adding 0.2 mL of the enzyme solution and reacting at 37 ° C. for 30 minutes, the reaction was stopped by adding a 15% acetic acid solution, and the absorbance at 410 nm was measured. In the above method, the amount of enzyme that liberates 1 μmol of p-nitroaniline per minute was defined as 1 unit.

3-3. Proteinase activity assay
To 0.9 mL of 0.6% azocasein (Sigma) aqueous solution, 0.3 mL of 0.1 M phosphate buffer (pH 7.0) was added, and 0.4 mL of enzyme solution was added. After reacting at 37 ° C. for 30 minutes, 2.4 mL of 10% trichloroacetic acid was added to stop the reaction. After filtration through Toyo Filter Paper No. 131, the absorbance at 400 nm was measured. The amount of enzyme that increases the absorbance by 1.0 per minute under the above conditions was defined as 1 unit.

4). Measurement of protein amount Using bovine serum albumin as a standard, measurement was performed using a protein assay reagent of Bio-Rad. During chromatography, the absorbance was measured at 280 nm and the protein was monitored.

5). Purification of peptidase
The culture supernatant obtained by jar fermenter culture cultured for 72 hours was concentrated with an ultrafiltration membrane (AIV-1010; Asahi Kasei Chemicals Corporation) and used for purification of the following peptidases.

5-1. Salting out Solid ammonium sulfate was added to and dissolved in the concentrated solution to a saturation concentration of 80. After standing at 5 ° C. for 20 hours, the crude enzyme was collected by filtration, dissolved in purified water, and dialyzed against tap water for 8 hours and against 0.01M phosphate buffer (pH 7.0) for 16 hours.

5-2. DEAE-Toyopearl 650 M column chromatography
The concentrated solution dialyzed against 0.01 M phosphate buffer (pH 7.0) was placed on a DEAE-Toyopearl 650 M column (4.0 × 50 cm) equilibrated with the same buffer, and the adsorbed fraction was linearly adjusted to the salt concentration. Raised (0-0.5M) and eluted (60 mL / h). The eluted active fractions were collected, ammonium sulfate was dissolved to a 20% solution concentration, and subjected to the next chromatography.

5-3. Butyl-Toyopearl 650M column chromatography
Place the active fraction on a Butyl-Toyopearl 650M column (4.0 x 30 cm) equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 20% ammonium sulfate. Was linearly lowered (20-0%) and eluted (50 mL / h). The eluted active fractions were collected, dialyzed against 0.01 M phosphate buffer (pH 7.0), and then subjected to the next chromatography.

5-4. Hydroxylapatite column chromatography
The obtained active fraction was placed on a column (4.5 x 30 cm) equilibrated with 0.01 M phosphate buffer (pH 7.0), and the phosphate concentration was increased linearly (10 to 250 mM). Fractions were eluted (20 mL / h). The eluted active fractions were collected, dialyzed against 0.02M phosphate buffer (pH 7.0) containing 0.5M NaCl, and then subjected to the next chromatography.

5-5. Toyopearl HW-55F column chromatography
The active fraction was placed on a column (2.2 × 90 cm) equilibrated with 0.02 M phosphate buffer (pH 7.0) containing 0.5 M NaCl, and gel filtration was performed at a flow rate of 15 mL / h. The eluted active fractions were collected and dialyzed against 0.01 M phosphate buffer (pH 7.0), and then used as a purified enzyme in the following experiments. Hereinafter, the purified carboxypeptidase is referred to as CP31-B, and the aminopeptidase is referred to as AP31-B.

6). Examination of characteristics of purified enzyme 6-1. SDS-PAGE
The purity and molecular weight of each purified peptidase were confirmed by SDS-PAGE. Phosphorylase b (97.2 kDa), bovine serum albumin (66.4 kDa), ovalbumin (45.0 kDa), carbonic acid dehydrogenase II (29.0 kDa), soybean trypsin inhibitor (21.0 kDa) and lysozyme (14.3 kDa) were used as molecular weight markers. After electrophoresis using C-PAGEL and Compact PAGE AE-7305 (Ato Co., Ltd.), the cells were stained with Coomassie Brilliant Blue.

6-2. Measurement of optimum pH and temperature
AP31-B was measured according to the activity measurement method (LAP method), and CP31-B was measured at the optimum pH by the CP method and at the optimum temperature by the ACP method.

6-3. Substrate specificity
AP31-B was measured according to the activity measurement method (LAP method) using amino acid-pNA (L-amio p-nitroanilide) as a substrate.

  CP31-B was measured by the CP method using various benzyloxycarbonyl dipeptides (Z-dipeptides) as substrates.

7). 7. Examination of peptidase degradation mode 7-1. Preparation of substrate solution
0.8 mMole of tetrapeptide (Taftusin; Peptide Institute, Inc.), tripeptide or dipeptide was dissolved in a solution of 2 mL of purified water added to 1 mL of 0.1 M McKilvine buffer (pH 5.0) (CP31-B above) in the case of). In the case of AP31-B, 0.1 M Tris buffer (pH 7.0) was used as the buffer.

7-2. reaction
1 mL of AP31-B solution (2.28 units) was added to each of the above substrate solutions, and a decomposition reaction was performed at 40 ° C. In the case of CP31-B, 1 mL (3.7 units of ACP activity) was added, and the decomposition reaction was performed at 50 ° C.

7-3. Qualitative analysis of free amino acid A small amount was sampled at each reaction time, and the enzyme was removed with a centrifuge tube with an ultrafiltration membrane (Spin-X UF, CORNING), and then free amino acid was confirmed by TLC. Use a silica gel 60F ₂₅₄ (Merck) as the TLC plate, butanol: acetic acid: water (4: 1: 2.5 (V / V)) as the developing solvent, and spray a 95% n-butanol solution of 0.2% ninhydrin. Color developed at ℃.

8). Effects of metal ions and enzyme inhibitors Carboxypeptidase activity (CP method) and aminopeptidase activity (LAP method) were measured in the presence of various metal ions and enzyme inhibitors (1 mM).

9. Bitter taste reduction test 1 (effect on bitter taste peptides)
9-1. Preparation of Bitter Peptide 10 mL of 0.1 M NaOH solution was added to 2 g of milk casein (manufactured by Wako Pure Chemical Industries, Ltd.) and dissolved by heating. After cooling, 10 mL of 0.1 M Tris buffer (pH 7.0) and 10 mL of purified water were added to adjust the pH to 7.0, and then adjusted to 50 mL with purified water. Furthermore, 100 mg of protin SD-AY10 (Amano Enzyme Co., Ltd.) was added, reacted at 50 ° C. for 3.5 hours, and then sterilized at 121 ° C. for 1 minute. In the CP31-B test, a bitter-tasting peptide was similarly prepared using Pro Leather FG-F (manufactured by Amano Enzyme Co., Ltd.) instead of Protin SD-AY10.

9-2. Preparation of bitterness standard solution Caffeine (Wako Pure Chemical Industries, Ltd.) 0 to 20 g / L of purified aqueous solutions of various concentrations were prepared. The threshold was approximately 200 mg / L.

9-3. Calculation of Peptide Degradation Rate Degradation rate was displayed as a ratio of formol nitrogen by formol titration method to total nitrogen by Kjeldahl method.

9-4. Bitterness reduction reaction 3 mL of the enzyme solution was added to 27 mL of the bitter peptide solution, reacted for 1, 3 or 5 hours, and the bitterness was measured by a sensory test. In the case of CP31-B, 3.22 units of ACP activity were added and reacted at 50 ° C. (pH 6.0). In the case of AP31-B, 6.81 units were added and reacted at 40 ° C. (pH 7.0).

10. Bitterness reduction test 2 (effect on yogurt)
10-1. Yogurt preparation
In the AP31-B addition test, 250 mL of milk (Meiji delicious milk, Meiji Co., Ltd.) and 0.5 g of inoculum (Yogurt's wish: Rizière Doole RD Co., Ltd.) and AP31-B (1.07, 2.15, or 4.30 units), respectively In addition, after leaving at 28 ° C. for 24 hours, it was left at 5 ° C. for 1 hour to perform sensory evaluation and measurement of glutamic acid production. In the CP31-B addition test, 0.5 g of inoculum (ROYAL KEFIR PRO; Royal Yuki Co., Ltd.) 0.5 g and peptidase (1.03, 3.09 or 5.15 units) were added to 250 mL of milk (Meiji delicious milk, Meiji Co., Ltd.), 25 After fermentation at 16 ° C. for 16 hours and then cooling at 5 ° C. for 1 hour, sensory evaluation and measurement of glutamic acid production were performed. For the measurement of the amount of glutamic acid, Yamasa L-glutamic acid measurement kit II (Yamasa Shoyu Co., Ltd.) was used.

10-2. Sensory evaluation of yogurt Kashiyama Jogakuen University university students (21 ± 1 years old) tasted by 10 students, ranked by evaluation method by rank method, Kramer test (Kramer, A. (1960). Rapid method for determination Significance of difference from ranking. Food Technol., 14,576-582.

B. Results and Discussion 1. Identification of 31-B Strain Yeast 31-B isolated from Nepenthes digestion solution was subjected to 26S rDNA-D1 / D2 nucleotide sequence analysis at Techno Suruga (Shizuoka City, Japan). The base sequence of 26S rDNA-D1 / D2 showed 100% homology to the sequence of Pseudozyma hubeiensis CBS 10077 ^T (DQ008953). Strain 31-B grew aerobically on yeast extract-malt extract agar medium (Becton Dickinson) (25 ° C., 7 days). Oviform or oval budding was observed, and no reproductive organs were observed after 3 weeks of observation. The colonies were creamy with white to yellowish white and somewhat mucous and convex on the surface. These characteristics correspond to those of Pseudozyma huveiensis (Wang, QM., Jia, JH. And Bai, FY. (2006) Int. J. Syst. Evol. Microbiol., 56, 289-293.). From the above results, the 31-B strain was identified as Pseudozuma hubeiensis.

2. Cultivation of 31-B strain Both peptidases produced up to 72 hours of culture and could be recovered stably (data not shown).

3. Purification of peptidase From the culture broth cultured for 72 hours with medium composition C, insoluble matters including 31-B strain cells were removed by centrifugation (6000 rpm, 4 ° C., 10 min). The supernatant was concentrated about 5 times with an ultrafiltration membrane (AIV 1010; Asahi Kasei Chemicals Corporation). Add ammonium sulfate as a solid to the concentrate and dissolve to a saturation concentration of 80, leave it at 5 ° C for 20 hours, collect the precipitate, dissolve in purified water, desalinate by dialysis, and dialyse to 0.01M phosphate buffer. And subjected to DEAE-Toyopearl 650M chromatography equilibrated with the same buffer. As a result of DEAE-Toyopearl 650M chromatography (FIG. 1), aminopeptidase and carboxypeptidase were fractionated into respective active fractions, and the subsequent purification was carried out for each peptidase.

3-1. Purification of aminopeptidase Table 2 shows the results of purification of aminopeptidase.

  Proteinase was not detected in the culture supernatant of 31-B strain. Also, aminopeptidase isozymes were not observed throughout the purification process. Finally, aminopeptidase (AP31-B) could be purified to a specific activity of about 107 times with a yield of 8.0%.

カルボキシペプチダーゼ（CP31-B）は、収率7％で比活性は約250倍に精製できた。また、精製過程を通して、カルボキシペプチダーゼのアイソザイムは認められなかった。尚、試薬カルボキシペプチダーゼ（CPY）との活性比較を表４に示す。

＊オリエンタル酵母工業（株）製 3-2. Purification of carboxypeptidase Table 3 shows the results of purification of carboxypeptidase.

Carboxypeptidase (CP31-B) was purified at a yield of 7% and specific activity about 250 times. Also, carboxypeptidase isozymes were not observed throughout the purification process. The activity comparison with the reagent carboxypeptidase (CPY) is shown in Table 4.

* Oriental Yeast Industry Co., Ltd.

4). SDS-PAGE of purified enzyme
The results of SDS-PAGE are shown in FIG. 2 (AP31-B) and FIG. 3 (CP31-B). Both enzymes were purified as a single band by SDS-PAGE. Regarding the molecular weight, AP31-B had a molecular weight of 75.3 kDa and CP31-B had a molecular weight of 70.5 kDa.

5). Enzymological examination of purified enzyme 5-1. Temperature and activity FIG. 4 shows the measurement results of carboxypeptidase (CP31-B). Carboxypeptidase activity was measured under the condition of pH 5.0 according to the ACP activity measurement method. The optimum temperature was 50 ° C. On the other hand, the result of aminopeptidase (AP31-B) is shown in FIG. The optimum temperature was 40 ° C. (pH 8.0).

5-2. pH and activity Carboxypeptidase was measured by the CP method at 40 ° C. (FIG. 6). The optimum pH was 5. The optimum pH of aminopeptidase was 8 (37 ° C.) (FIG. 7).

5-3. Effect of metal ion and enzyme inhibitor on enzyme activity In the measurement of aminopeptidase activity, the reaction was carried out at pH 8.0, 40 ° C. for 30 minutes, and a metal salt or enzyme inhibitor at a concentration of 1 mM was added to the reaction system. On the other hand, carboxypeptidase activity was measured by a CP activity measurement method (pH 5.0, 30 minutes, 50 ° C.), and a metal salt or enzyme inhibitor having a concentration of 1 mM was added to the reaction system. The results for aminopeptidase are shown in Table 5.

Aminopeptidase is completely inhibited by zinc ions (Zn ²⁺ ), and is also activated by divalent iron ions (Fe ²⁺ ), copper ions (Cu ²⁺ ) and trivalent iron ions (Fe ³⁺ ). Inhibited. The activity was also inhibited by SDS, EDTA and PCMB. The activity is not inhibited by E-64, Pepstatin A and Petabloc. On the other hand, potassium ion (K ⁺ ) promoted activation.

The results for carboxypeptidase are shown in Table 6.

The activity of carboxypeptidase was inhibited by potassium ion (K ⁺ ), manganese ion (Mn ²⁺ ) and divalent iron ion (Fe ²⁺ ). The activity was also inhibited by SDS and PCMB.

5-4. Substrate specificity Aminopeptidase activity was measured according to the LAP method. Carboxypeptidase activity was measured by CP method. The measurement results (substrate specificity) of aminopeptidase are shown in Table 7.

  Aminopeptidase (AP31-B) acted on N-terminal leucine, arginine, methionine and alanine. On the other hand, it slightly affected glycine and proline at the N-terminal of the peptide chain. For oligopeptides, aminopeptidase (AP31-B) released only threonine from tuftsin (Thr-Lys-Pro-Arg) and only leucine from tripeptide (Leu-Ala-Pro). . Leucine and alanine were released from the tripeptide (Leu-Leu-Ala). From these results, it was speculated that aminopeptidase (AP31-B) did not act on the N-terminal amino acid bound to proline. Thus, aminopeptidase (AP31-B) showed similar substrate specificity to the previously reported aminopeptidase.

The measurement results of carboxypeptidase are shown in Table 8.

  Carboxypeptidase (CP31-B) acted on C-terminal leucine and phenylalanine. On the other hand, the effects on the C-terminal alanine, proline and tyrosine of the peptide chain were negligible. Thus, the substrate specificity of carboxypeptidase (CP31-B) generally tended to resemble CPY.

6). Bitter Peptide Reduction by Peptidase The prepared bitter peptide has a degradation rate of 8.5% (used for the AP31-B test) or 11% (test used for the CP31-B), both of which have a bitterness equivalent to 20 g / L of caffeine. Presented. The degradation process and the process of lowering bitterness associated therewith are shown in FIGS. Peptide bitterness was reduced to less than 1/10 in all cases.

色、香り；数値が低い方が好まれる、酸味、甘味；数値が低い方が強く感じる、苦味；数値が低い方が弱く感じる、うま味；数値が低い方が強く感じる、総合；数値が低い方が好まれる。順位総合で１７−３３を超える範囲で危険率５％有意差あり、１５−３５を超える範囲で危険率１％有意差あり。**は、危険率１％有意差あり。 7). Effect of peptidase addition during yogurt preparation Table 9 and Table 10 show the evaluation results for aminopeptidase (AP31-B).

Color, fragrance; lower value is preferred, acidity, sweetness; lower value is stronger, bitterness; lower value is weaker, umami; lower value is stronger, overall; lower value Is preferred. There is a 5% significant difference in risk rate in the range exceeding 17-33 in the overall ranking, and a 1% significant difference in risk rate in the range exceeding 15-35. ** indicates a significant difference of 1% in the risk rate.

  Yogurt added with aminopeptidase (AP31-B) tends to have a higher evaluation of bitterness and umami taste than control (control), and the bitterness of control (control) tends to decrease. there were. Yogurt added with aminopeptidase (AP31-B) tended to be preferred in the overall evaluation.

色；数値が低い方が好まれる、酸味、甘味；数値が低い方が強く感じる、苦味、塩味；数値が低い方が弱く感じる、うま味；数値が低い方が強く感じる、総合；数値が低い方が好まれる。順位総合で１７−３３を超える範囲で危険率５％有意差あり、１５−３５を超える範囲で危険率１％有意差あり。**は、危険率１％有意差あり、*は、危険率５％有意差あり。 The evaluation results for carboxypeptidase (CP31-B) are shown in Tables 11 and 12.

Color: Lower value is preferred, acidity, sweetness; lower value is stronger, bitterness, saltiness; lower value is weaker, umami; lower value is stronger, overall; lower value Is preferred. There is a 5% significant difference in risk rate in the range exceeding 17-33 in the overall ranking, and a 1% significant difference in risk rate in the range exceeding 15-35. ** has a significant difference of 1%, and * has a significant difference of 5%.

  Yogurt (1.03 and 3.09 U / 250 mL milk) supplemented with carboxypeptidase (CP31-B) tended to be highly evaluated in terms of color and sweetness. Also, yogurt (3.09U / 250mL milk) favored bitterness and saltiness. At any added amount, the bitter taste of the control (control) tended to be reduced, and the evaluation was higher than the control (control) in terms of overall score.

<Application to food processing>
Several enzyme agents have already been registered as food additive enzymes as peptidases, most of which are enzyme agents mainly composed of proteinases. In protein food processing, proteinase is mainly used, but if it is used for the purpose of liberating only amino acids, for example, for the purpose of eliminating bitterness of peptides, proteinase will be affected. Peptidase agents that do not contain are useful. Through the above experiments, we succeeded in purifying the peptidase produced by Pseudozyma Hubeiensis 31-B and clarified its properties. In the following, we decided to examine the usefulness of peptidase produced by 31-B strain in the food processing field.

A. Materials and Methods Enzyme Pseudozyma / Fubeyensis strain 31-B cultured, centrifuged, salted out, and dissolved crude enzyme solution (before the peptidase purification step in the above experiment) was used as a sample (hereinafter referred to as “PP31”). We used for examination. For comparison, Rhizopus-origin peptidase R (manufactured by Amano Enzyme) and Aspergillus-origin proteax (manufactured by Amano Enzyme), which are commercially available food additive enzyme agents, were used.

2. Bitter taste reduction test of bitter taste peptide Although it implemented according to the said experiment, 100 mg of pro leather FG-F (made by Amano Enzyme Co., Ltd.) which is a Bacillus subtilis protease was used for peptide preparation. The degradation rate of milk casein was 15-17%. In the bitterness reduction reaction, 3 mL of enzyme solution (5 units of ACP activity) was added to 27 mL of the bitter peptide solution and reacted at 37 ° C. The bitterness was evaluated by 3 panelists from Ulsan Jogakuen University (21 ± 1 years old) and compared with caffeine solutions of various concentrations.

B. Results and discussion Enzyme activity measurement results The results are shown in Table 13.

  The proteinase activity of PP31 (salting out solution) was substantially zero. That is, it became clear that there was substantially no proteinase to contaminate.

2. 2. Examination of enzyme properties 2-1. Effect of pH on aminopeptidase activity The experimental results are shown in FIG.

2-2. Effect of temperature on peptidase activity 2-2-1. The results of the aminopeptidase experiment are shown in FIG.

2-2-2. Carboxypeptidase Experimental results are shown in FIG.

3. 3. Bitter taste reduction test of bitter peptide by enzyme agent 3-1. Bitter taste reduction effect by PP31
FIG. 13 shows the experimental results when the reaction is performed at pH 7.0. The degradation rate increased by 7.40% and the bitterness was reduced to 1/5 or less. FIG. 14 shows the experimental results when the reaction is performed at pH 5.0. The degradation rate increased by 12.06% and the bitterness was less than half. Peptidase R increased the degradation rate by 17.4%, but the bitterness rather increased after 3 hours (data not shown). For proteax, the degradation rate increased by 13.4%, but the bitterness decreased slightly (data not shown).

  A novel peptidase derived from Pseudozyma huveiensis is provided. The peptidase of the present invention can be used, for example, for the production and processing of various foods.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (14)

Peptidases with the following enzymatic chemistry:
(1) Action It catalyzes a reaction that liberates the N-terminal amino acid of the peptide chain;
(2) Molecular weight The molecular weight by SDS-PAGE is about 75.3 kDa;
(3) Substrate specificity Releases leucine, arginine, methionine, and alanine at the N-terminus of the peptide chain. The effect on N-terminal glycine and proline of the peptide chain is weak. Does not affect the N-terminal amino acid bound to proline;
(4) optimum pH about 8;
(5) Optimum temperature about 40 ℃. Peptidases with the following enzymatic chemistry:
(1) Action It catalyzes a reaction that liberates the C-terminal amino acid of the peptide chain;
(2) Molecular weight The molecular weight by SDS-PAGE is about 70.5 kDa;
(3) Substrate specificity It works well on leucine and phenylalanine at the C-terminal of the peptide chain. Weak effect on alanine, proline and tyrosine at the C-terminus of the peptide chain;
(4) optimum pH about 5;
(5) Optimum temperature about 50 ℃.   The peptidase according to claim 1 or 2, wherein the peptidase is derived from Pseudozyma huveiensis.   The peptidase according to claim 3, wherein Pseudozyma hubeiensis is Pseudozyma hubeiensis 31-B strain.   The enzyme agent which uses the peptidase as described in any one of Claims 1-4 as an active ingredient.   An enzyme agent comprising the peptidase according to claim 1 and the peptidase according to claim 2. A method for producing a peptidase comprising the following steps (1) and (2):
(1) culturing Pseudozyma huveiensis;
(2) A step of recovering peptidase from the culture solution after culturing.   The production method according to claim 7, wherein Pseudozyma Hubeiensis is Pseudozyma Hubeiensis 31-B strain.   The production method according to claim 7 or 8, wherein aminopeptidase and carboxypeptidase are recovered as the peptidase in step (2).   The production method according to claim 7 or 8, wherein aminopeptidase or carboxypeptidase is recovered as the peptidase in step (2).   Pseudozyma hubeiensis 31-B strain identified by accession number NITE P-02047.   In the production process of food, the peptidase according to any one of claims 1 to 4 or the enzyme agent according to claim 5 or 6 is allowed to act on a raw material or intermediate processed product containing protein or peptide. A method for producing or processing food, characterized in that   The method of claim 12, wherein the food product is a dairy product.   The foodstuff which bitterness reduced by the effect | action of the peptidase as described in any one of Claims 1-4, or the enzyme agent of Claim 5 or 6.

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